Analysis and optimization of the cationic lipid component of a lipid/peptide vector formulation for enhanced transfection in vitro and in vivo

We have previously described a lipopolyplex formulation comprising a mixture of a cationic peptide with an integrin-targeting motif (K16GACRRETAWACG) and Lipofectin, a liposome consisting of DOTMA and DOPE in a 1:1 ratio. The high transfection efficiency of the mixture involved a synergistic interaction between the lipid/peptide components. The aim of this study was to substitute the lipid component of the lipopolyplex to optimize transfection further and to seek information on the structure-activity relationship of the lipids in the lipopolyplex. Symmetrical cationic lipids with diether linkages that varied in alkyl chain length were formulated into liposomes and then incorporated into a lipopolyplex by mixing with an integrin-targeting peptide and plasmid DNA. Luciferase transfections were performed of airway epithelial cells and fibroblasts in vitro and murine lung airways in vivo. The biophysical properties of lipid structures and liposome formulations and their potential effects on bilayer membrane fluidity were determined by differential scanning calorimetry and calcein-release assays. Shortening the alkyl tail from C18 to C16 or C14 enhanced lipopolyplex and lipoplex transfection in vitro but with differing effects. The addition of DOPE enhanced transfection when formulated into liposomes with saturated lipids but was more variable in its effects with unsaturated lipids. A substantial improvement in transfection efficacy was seen in murine lung transfection with unsaturated lipids with 16 carbon alkyl tails. The optimal liposome components of lipopolyplex and lipoplex vary and represent a likely compromise between their differing structural and functional requirements for complex formation and endosomal membrane destabilization.     

Polyamines Are Not Required for Aerobic Growth of Escherichia coli: Preparation of a Strain with Deletions in All of the Genes for Polyamine Biosynthesis

A strain of Escherichia coli was constructed in which all of the genes involved in polyamine biosynthesis—speA (arginine decarboxylase), speB (agmatine ureohydrolase), speC (ornithine decarboxylase), spe D (adenosylmethionine decarboxylase), speE (spermidine synthase), speF (inducible ornithine decarboxylase), cadA (lysine decarboxylase), and ldcC (lysine decarboxylase)—had been deleted. Despite the complete absence of all of the polyamines, the strain grew indefinitely in air in amine-free medium, albeit at a slightly (ca. 40 to 50%) reduced growth rate. Even though this strain grew well in the absence of the amines in air, it was still sensitive to oxygen stress in the absence of added spermidine. In contrast to the ability to grow in air in the absence of polyamines, this strain, surprisingly, showed a requirement for polyamines for growth under strictly anaerobic conditions.Polyamines are highly abundant in essentially all organisms, ranging from bacteria to humans, and there have been a large number of studies from this and many other laboratories reporting a variety of phenotypic effects resulting from changes in the concentration of polyamines in both in vitro and in vivo experiments. In particular, polyamines have been associated with such biological processes as nucleic acid and protein biosynthesis and structure, cell growth, and differentiation (reviewed in references 5, 22, and 23). Therefore, it was surprising that, in our earlier studies (24), we found that a mutant of Escherichia coli that had mutations in the genes for the biosynthesis of the polyamines (ΔspeA, ΔspeB, ΔspeC, ΔspeD, ΔspeE, and cadA) still grew indefinitely in a polyamine-free medium, albeit at a decreased growth rate (ca. 30% of the normal growth rate).The strain used in our previous studies still had trace amounts of putrescine and significant amounts of cadaverine. To study whether these small amounts of amines could account for the slow growth of these strains, we have now constructed a new strain that is completely deficient in these amines by including deletions of cadA (inducible lysine decarboxylase), ldcC (constitutive lysine decarboxylase), and speF (inducible ornithine decarboxylase) to the strain described above. We found that this strain which is completely deficient in all of the amines still grows well (40 to 50% of normal growth rate) in purified medium in air. This indicates that, at least for this organism, the various physiological functions attributed to polyamines are not required for growth in air. In contrast, we have found that polyamines are required for growth of this strain in 95% oxygen and under anaerobic conditions.     

Constituents of antibacterial extract of Caesalpinia paraguariensis Burk

The Argentinean legume Caesalpinia paraguariensis Burk. (Fabaceae) was selected for further fractionation work based on the strong antimicrobial activity of its CH2Cl2-MeOH (1:1 v/v) extract against a host of clinically significant microorganisms, including antibiotic resistant strains. 1D and 2D NMR enabled the identification of the novel benzoxecin derivative caesalpinol along with the known compounds bilobetin, stigma-5-en-3-O-beta-6'-stearoylglucopyranoside, stigma-5-en-3-beta-6'-palmitoylglucopyranoside, stigma-5-en-3-beta-glucopyranoside, oleanolic acid, 3-O-(E)-hydroxycinnamoyl oleanolic acid, betulinic acid, 3-O-(E)-hydroxycinnamoyl betulinic acid, and lupeol from the active fractions. Oleanolic acid was found active against Bacillus subtilis and both methicillin-sensitive and -resistant Staphylococcus aureus with MICs of 8 (17.5 microM), 8 and 64 (140 microM) microg/ml, respectively. The rest of the compounds, however, did not show activity.     

Finishing a whole-genome shotgun: release 3 of the Drosophila melanogaster euchromatic genome sequence

Background

The Drosophila melanogaster genome was the first metazoan genome to have been sequenced by the whole-genome shotgun (WGS) method. Two issues relating to this achievement were widely debated in the genomics community: how correct is the sequence with respect to base-pair (bp) accuracy and frequency of assembly errors? And, how difficult is it to bring a WGS sequence to the accepted standard for finished sequence? We are now in a position to answer these questions.

Results

Our finishing process was designed to close gaps, improve sequence quality and validate the assembly. Sequence traces derived from the WGS and draft sequencing of individual bacterial artificial chromosomes (BACs) were assembled into BAC-sized segments. These segments were brought to high quality, and then joined to constitute the sequence of each chromosome arm. Overall assembly was verified by comparison to a physical map of fingerprinted BAC clones. In the current version of the 116.9 Mb euchromatic genome, called Release 3, the six euchromatic chromosome arms are represented by 13 scaffolds with a total of 37 sequence gaps. We compared Release 3 to Release 2; in autosomal regions of unique sequence, the error rate of Release 2 was one in 20,000 bp.

Conclusions

The WGS strategy can efficiently produce a high-quality sequence of a metazoan genome while generating the reagents required for sequence finishing. However, the initial method of repeat assembly was flawed. The sequence we report here, Release 3, is a reliable resource for molecular genetic experimentation and computational analysis.     

Effects of Differently Composed Feeds and Physical Stress on Plasma Gastrin Concentration in Horses

Plasma gastrin concentrations were determined in 6 Standardbreds (4 geldings and 2 mares) after 3 different meals consisting of unlimited amounts of hay (8-9 kg per horse), a restricted amount of hay (0.6 kg/100 kg body-weight) and grain (0.2 kg/100 kg body-weight) in combination or of grain alone (0.2 kg/100 kg body-weight). In another series of experiments the possible role of gastrin as a stress hormone was investigated. Plasma gastrin and Cortisol concentrations were determined during fasting and compared with concentrations during hay feeding. In addition, gastrin and Cortisol concentrations were determined before, during and after 2 kinds of physical exercise on a treadmill. Meal stimulation significantly increased the plasma gastrin concentration, irrespective of the meal composition. An immediate and large increase in plasma gastrin concentration was found when voluminous meals were given, whereas a small meal evoked a later onset of gastrin release, suggesting that gastric distention plays an important role in inducing gastrin release during a meal. Meals consisting of grain seem to evoke a slower onset and then a more prolonged gastrin response than a hay meal, possibly due to different emptying rates of the stomach. Nervous excitation may play a minor role in the activation of gastrin release in horses. No experimental support was obtained for the idea that gastrin acts as a stress hormone in the horse. kw|Keywords|k]Cortisol; k]exercise; k]feeding; k]fasting     

SPE1 and SPE2: two essential genes in the biosynthesis of polyamines that modulate +1 ribosomal frameshifting in Saccharomyces cerevisiae.

We previously showed that a mutant of Saccharomyces cerevisiae, which cannot make spermidine as a result of a deletion in the SPE2 gene (spe2 delta), exhibits a marked elevation in +1 ribosomal frameshifting efficiency in response to the Ty1 frameshift sequence, CUU AGG C. In the present study, we found that spermidine deprivation alone does not result in increased +1 ribosomal frameshifting efficiency. The high level of +1 ribosomal frameshifting efficiency in spe2 delta cells is the result of the combined effects of both spermidine deprivation and the large increase in the level of intracellular putrescine resulting from the derepression of the gene for ornithine decarboxylase (SPE1) in spermidine-deficient strains.     

Inhibitory Effect of Solar Radiation on Amino Acid Uptake in Chesapeake Bay Bacteria

The effect of solar radiation on a natural bacterial population from the Chesapeake Bay was evaluated from measured changes in numbers of organisms engaged in amino acid uptake. From July through May, freshly collected water samples were exposed in quartz containers to 3.5 h of total sunlight both with and without UV-absorbing filters. Water samples were subsequently incubated with tritiated amino acids, and the uptake-active bacteria were assayed by microauto-radiography-epifluorescence microscopy. The survival index, defined as the fraction of the uptake-active population that remained active after the exposure to sunlight, ranged from 0.93 to 0.20. Decreased survival was correlated with increased solar intensity. The inhibition of amino acid uptake was attributed not only to the UV-B component of the solar spectrum (280 to 320 nm), but also to longer UV and visible wavelengths.     

Identification of pyruvate in S-adenosylmethionine decarboxylase from rat liver

S-Adenosylmethionine decarboxylase has been purified to homogeneity (26,000-fold) from rat liver. The enzyme has a molecular weight of 155,000 and a subunit molecular weight of 42,000. One mole of covalently bound pyruvate was found to be present per mole of enzyme subunit. This is the first mammalian enzyme found to contain covalently linked pyruvate.     

Rapid determination of dipicolinic acid in the spores of Clostridium species by gas-liquid chromatography.

A gas-liquid chromatographic procedure has been developed to quantitate dipicolinic acid in bacterial spores. The culture, washed from a plate, was hydrolyzed with acid containing the internal standard, pyridine-2,4-dicarboxylate, and then extracted into methyl isobutyl ketone. The internal standard and dipicolinic acid were then extracted into a small volume of trimethylphenylammonium hydroxide. Injection of the resultant quaternary ammonium salts into a gas chromatograph yielded, via thermal decomposition, the methyl ester derivatives of the dipicolinic acid and the internal standard. The amount of dipicolinic acid in the sample was determined from a standard curve. The method was sensitive to 100 ng of dipicolinic acid per sample and was 1,000 to 5,000 times more sensitive than the commonly used methods. Preparation of the sample required less than 1.5 h and less than 15 min of the analyst's time.